Disproportional enrichment of FoxP3+CD4+ regulatory T cells shapes a suppressive tumour microenvironment in head and neck squamous cell carcinoma

The efficacy of PD-1 inhibitors has fallen short in human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC), underscoring the clinical rel-evance of exploring the mechanism shaping treatment resistance. In this study, the suppressive tumour microenvironment of HNSCC was investigated to gain insights into anti-PD-1 resistance and conceive the better treatment option. IFN-γ antitumour


Disproportional enrichment of FoxP3 + CD4 + regulatory T cells shapes a suppressive tumour microenvironment in head and neck squamous cell carcinoma
Dear Editor, The efficacy of PD-1 inhibitors has fallen short in human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC), underscoring the clinical relevance of exploring the mechanism shaping treatment resistance. In this study, the suppressive tumour microenvironment of HNSCC was investigated to gain insights into anti-PD-1 resistance and conceive the better treatment option.
Based on transcriptomic data derived from the Cancer Genome Atlas (TCGA), 1 HNSCC had the highest frequency of the IFN-γ-dominant subtype, with over 70% of cases classified as IFN-γ-dominant ( Figure 1A). Since immune checkpoint blockade has shown modest efficacy in treating HNSCC, 2-5 we investigated the key mechanisms involved in shaping an immune-suppressive tumour microenvironment that counterbalances the active IFNγ signature. We reasoned that the robust adaptive antitumour immune response is actively suppressed by a subpopulation of tumour-infiltrating (TI) lymphocytes, and therefore investigated the expression of FoxP3, a specific marker for regulatory T cells (Tregs). 6 Intriguingly, FoxP3 expression was highest in the HNSCC cohort, along with the frequency of the IFN-γ-dominant subtype (Figures 1B and S1A), although the expression of CD4, CD8A and CD8B was intermediate in HNSCC (Figure S1B-D). Therefore, we focused on TI Tregs in HNSCC, which are hypothesised to underlie the key mechanisms involved in shaping an immunosuppressive microenvironment that results in checkpoint blockade resistance.
Transcriptomic analysis of HNSCC cohort from TCGA revealed that the expressions of FoxP3 and immune checkpoints were higher in tumour tissue than in normal adjacent tissue ( Figure 1C). Flow cytometry analysis of clinical samples of HNSCC patients confirmed that the frequency of Tregs was higher in tumour than in normal adjacent tissue or peripheral blood ( Figures 1D and S2 ingly, PD-1 + cells were more frequently observed among Tregs in tumour tissue ( Figure 1E). These PD-1 + TI Tregs expressed immune checkpoint receptors and Ki-67 more frequently than PD-1 − TI Tregs ( Figure 1F), suggesting that these Tregs impeded CD8 + T-cell functionality ( Figure S3).
Based on the previous findings of the prognostic implications of HPV status in HNSCC, 7 we further classified patients in terms of HPV status and differential T-cell infiltration. Consistent with the transcriptomic data ( Figure  S4), immunohistochemical analysis revealed that both CD8 + T cells and Tregs exhibited greater infiltration in HPV-positive than in HPV-negative HNSCC (Figures 2A  and S5), with an apparent positive correlation between the infiltration rates of CD8 + T cells and Tregs ( Figure 2B). HPV-positive and CD8 high HNSCC patients demonstrated more favorable outcomes than HPV-negative and CD8 low patients in terms of both DFS and OS in the overall population, whereas DFS and OS did not differ according to the abundance of TI FoxP3 + T cells ( Figures 2C and S6A). Next, we conducted subgroup analyses according to HPV positivity. We observed no association between CD8 + Tcell infiltration and outcome in the HPV-negative group. Interestingly, FoxP3 high patients exhibited better survival outcomes in terms of DFS and OS than FoxP3 low patients in the HPV-negative group; likewise, FoxP3/CD8 high patients had better prognoses than FoxP3/CD8 low patients in the HPV-negative group ( Figures 2D and S6B). In the HPVpositive group, CD8 high patients displayed better survival outcomes than CD8 low patients in terms of both DFS and OS, whereas differences between outcomes based on the abundance of TI Tregs were not evident. The FoxP3/CD8 high group showed poor clinical outcomes, which was contrary to observations in HPV-negative patients ( Figures 2E and S6C). Collectively, these results indicated that the balance between TI CD8 + T cells and Tregs differentially impacted individual survival according to HPV status.
To evaluate the differences in the properties between TI Tregs in HPV-positive and HPV-negative HNSCC, we first analysed Cibersort for TCGA HNSCC cohort, which revealed that the relative fraction of Tregs was larger in HPV-positive than in HPV-negative HNSCC ( Figure 2F). Moreover, gene set enrichment analysis revealed that the expression of genes downstream of FoxP3 was significantly upregulated in HPV-positive compared to that in HPV-negative HNSCC ( Figure 2G). To address whether Tregs shape the suppressive tumour microenvironment in a quantitative or qualitative manner, we assessed the frequency and characteristics of TI Tregs using per-cellbased analysis. Indeed, FoxP3 + Tregs were significantly enriched in HPV-positive than in HPV-negative HNSCC ( Figure 2H), albeit the expression of immune checkpoint inhibitory receptors in TI Tregs did not differ significantly between two groups ( Figure 2I). In contrast, the expression of Ki-67 was slightly higher in TI Tregs from HPV-positive than HPV-negative tumours ( Figure 2I). This trend was maintained when we analysed the frequency of immune checkpoint receptors co-expressing in PD-1 + cells among TI Tregs ( Figure S7).
Next, we investigated TCGA HNSCC data to identify the expression of upstream regulators during Treg differentiation. The expression of TGFB1 was higher in HPVnegative HNSCC, whereas that of TGFB2, TGFB3 and IL10 did not differ based on HPV status ( Figure 3A). In contrast, the expression of IDO1 and IDO2 was highly upregulated in HPV-positive compared to that in HPVnegative HNSCC ( Figure 3A). Furthermore, the correlation between the expression of IDO1 or IDO2 and the relative frequency of Tregs among CD4 + T cells was stronger in HPV-positive than in HPV-negative HNSCC ( Figure 3B), suggesting that IDO pathway activation can be one of the key mechanisms involved in Treg induction and maintenance of the HPV-positive HNSCC tumour microenvironment. We further confirmed via single-cell transcriptomic data analysis that IDO was highly expressed in dendritic cells from the tumour microenvironment ( Figure S8A), 8 as well as in CD14 + HLA-DR + CD11c + mature dendritic cells from PBMCs upon stimulation ( Figure S8B and C). Supporting the aforementioned findings, promising clinical activity was observed in a patient with HPV-positive HNSCC in an ongoing study of combined treatment with the IDO inhibitor epacadostat and the PD-1 inhibitor pembrolizumab ( Figure 3C and D).
In conclusion, we identified that Tregs hamper active antitumour immunity in HNSCC, underlying anti-PD-1 resistance. In particular, activation of the IDO pathway contributes to the enrichment of Tregs in HPV-positive HNSCC. A combined PD-1 and IDO blockade elicited a consequential response in HPV-positive HNSCC. Exploiting strategies to target Tregs or the IDO pathway would benefit patients with HPV-positive HNSCC.

A C K N O W L E D G E M E N T S
The authors would like to thank the patients and their families who participated in this study. This study was supported by the National Research Foundation (NRF) grants funded by the Korea government (NRF-2019M3A9B6065231, NRF-2021R1A2C2094629 and 2017M3A9E9072669 to HRK; 2017R1A5A1014560 and 2019M3A9B6065221 to SJH; NRF-2021R1I1A1A01059271 to CGK). This study was also supported by a Severance Hospital Research fund for Clinical excellence (C-2021-0016) and YUHAN research grant to HRK, the Korean Heath Technology R&D Project (HV20C0144 to SJH) through the Korean Health Industry Development Insti-  A patient with oropharyngeal cancer with lung metastasis was enrolled in prospective trials (NCT03358472) and achieved partial response with more than 70% tumour reduction. This patient was progression-free for 134 weeks, even after the scheduled discontinuation of epacadostat and pembrolizumab after completion of 35 cycles. (C) Representative CT scan images and (D) the summation of target lesion showing the reduction of tumour lesion. During the overall treatment course, the toxicity profiles of the combined IDO and PD-1 inhibitors were manageable, without any dose interruption or discontinuation required. ****p < .0001. Statistical analyses were performed (A) using unpaired Student's t test and (B) using Pearson's correlation test